Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation.
(41/1207)
The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression. (+info)
cAMP-induced phosphorylation and inhibition of Na(+)/H(+) exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor.
(42/1207)
The members of the regulatory factor (RF) gene family, Na(+)/H(+) exchanger (NHE)-RF and NHE3 kinase A regulatory factor (E3KARP) are necessary for cAMP to inhibit the epithelial brush border NHE isoform 3 (NHE3). The mechanism of their action was studied using PS120 fibroblasts stably transfected with rabbit NHE3 and wild type rabbit NHE-RF or wild type human E3KARP. 8-Bromo-cAMP (8-Br-cAMP) had no effect on Na(+)/H(+) exchange activity in cells expressing NHE3 alone. In contrast, in cells co-expressing NHE-RF, 8-Br-cAMP inhibited NHE3 by 39%. In vivo phosphorylation of NHE3 demonstrated that cAMP increased phosphorylation in two chymotrypsin-generated phosphopeptides of NHE3 in cells containing NHE-RF or E3KARP but not in cells lacking these proteins. The requirement for phosphorylation of NHE-RF in this cAMP-induced inhibition of NHE3 was examined by studying a mutant NHE-RF in which serines 287, 289, and 290 were mutated to alanines. Wild type NHE-RF was a phosphorylated protein under basal conditions, but treatment with 8-Br-cAMP did not alter its phosphorylation. Mutant NHE-RF was not phosphorylated either under basal conditions or after 8-Br-cAMP. 8-Br-cAMP inhibited NHE3 similarly in PS120/NHE3 cells containing wild type or mutant NHE-RF. NHE-RF and NHE3 co-precipitated and did so similarly with and without cAMP. Mutant NHE-RF also similarly immunoprecipitated NHE3 in the presence and absence of 8-Br-cAMP. This study shows that members of the regulatory factor gene family, NHE-RF and E3KARP, are necessary for cAMP inhibition of NHE3 by allowing NHE3 to be phosphorylated. This inhibition is not dependent on the phosphorylation of NHE-RF. (+info)
CCAAT/enhancer-binding proteins are mediators in the protein kinase A-dependent activation of the decidual prolactin promoter.
(43/1207)
In the course of decidualization, human endometrial stromal cells (ESC) activate the alternative upstream promoter of the decidual prolactin (dPRL) gene. The dPRL promoter is induced by the protein kinase A pathway in a delayed fashion via the region -332/-270 which contains two overlapping consensus binding sequences, B and D, for CCAAT/enhancer-binding proteins (C/EBP). Here we show that sites B and D both bind C/EBPbeta and -delta from ESC nuclear extracts. When decidualization of cultured ESC was induced by treatment with 8-Br-cAMP, complex formation on sites B and D was enhanced. Western blot analysis revealed an elevation of both C/EBPbeta isoforms, liver-enriched activator protein and liver-enriched inhibitory protein, with a delayed onset between 8 and 24 h of cAMP treatment, while C/EBPdelta expression remained unaffected. Cyclic AMP-mediated activation of dPRL promoter construct dPRL-332/luc3 was abrogated by mutation of sites B and D at -310/-285. An expression vector for liver-enriched activator protein potently induced transcription of dPRL-332/luc3 and further enhanced cAMP-mediated induction, while liver-enriched inhibitory protein expression vector abolished the cAMP response, implying that C/EBPs serve as mediators in the delayed cAMP signal transduction to the dPRL promoter. The ratio between activating and repressing isoforms is likely to dictate the transcriptional output. (+info)
The decidualizing effect of progesterone may involve direct transcriptional activation of corticotrophin-releasing hormone from human endometrial stromal cells.
(44/1207)
The hypothalamic neuropeptide corticotrophin-releasing hormone (CRH) is also produced by human endometrial cells and is directly involved in the decidualization process as a paracrine inducer. The aim of the present work was to examine the effect of progesterone, the main decidualizing factor, on endometrial CRH, in primary cultures of human endometrial stromal cells. The effect of progesterone was examined by measuring the effects of medroxyprogesterone acetate (MPA) on (i) the concentration of immunoreactive CRH in isolated human endometrial stromal cells and (ii) the activity of the CRH promoter in human endometrial stromal cells transfected with a 0.9 kb fragment of the 5'-flanking region of the human CRH gene coupled to luciferase reporter. The data show that MPA increased the production and secretion of immunoreactive CRH from stromal cells and induced the activity of the CRH promoter, both in a dose-dependent manner. These effects were partially reversed by a molar excess of the antiprogestin RU 486 and were completely abolished in the presence of 100 nmol/l of the cAMP inhibitor, Rp-cAMP. The effect of progesterone on the CRH promoter requires the existence of an intact CRH sequence since experiments carried out with a deleted palindromic cAMP response element (CRE: 5'-TGACGTCA) at -224 bp of the CRH promoter resulted in a complete loss of MPA effect. In conclusion, these data provide evidence that progesterone induces the transcription of CRH gene in human endometrial stroma. This effect coupled with the decidualizing properties of progesterone and CRH may indicate that progesterone and CRH form a decidualizing local pathway within the human endometrium. (+info)
High cAMP in spores of Dictyostelium discoideum: association with spore dormancy and inhibition of germination.
(45/1207)
Signalling mechanisms involving cAMP have a well-documented role in the coordination of multicellular development and differentiation leading to spore formation in the social amoeba, Dictyostelium discoideum. The involvement of cAMP in the poorly understood developmental stages of spore dormancy and germination have been investigated in this study. Dormant spores contained up to 11-fold more cAMP than nascent amoebae. The spore cAMP levels were not constant, but typically underwent a surge at 14-18 d when spores acquired the ability to germinate spontaneously. The high cAMP levels decreased only during successful spore germination, i.e. emergence of nascent amoebae. The temporal pattern of cAMP decrease was complex and unique to the method of spore activation, supporting our hypothesis that exogenously (e.g. heat) activated and autoactivated spores germinate by different mechanisms. During heat-induced activation, transcription of acg (a gene encoding adenylyl cyclase associated with germination) correlated well with spore cAMP content. Young wild-type spores, incapable of spontaneous germination, maintained a uniformly high cAMP level, and spore cAMP levels also remained high if germination was inhibited. When activated spores were deactivated by applying increased osmotic pressure, cAMP concentrations rose and ultimately levelled off at the high levels typical of dormant spores. The correlation between high cAMP and failure to germinate was also evident when autoactivation was inhibited by the cAMP analogue, 8-bromo-cAMP. Also, spores from a strain (HTY217) with unrestrained protein kinase A activity were incapable of spontaneous germination. Overall, our experiments provide evidence for continued cAMP signalling in spores up to 18 d after sporulation and for linkages between elevated cAMP, spore deactivation and inhibition of spontaneous germination. (+info)
Protein kinase A is required for chromosomal DNA replication.
(46/1207)
Passage through mitosis resets cells for a new round of chromosomal DNA replication [1]. In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle [1]. The nuclear membrane also contributes to limit genome replication to once per cell cycle [2]. Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk [3] [4]. Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase. (+info)
Interaction of Shiga toxins with human brain microvascular endothelial cells: cytokines as sensitizing agents.
(47/1207)
Neurologic abnormalities are among the most serious extraintestinal complications of infection with Shiga toxin (Stx)-producing bacteria. Histopathologic examination of tissues from patients with extraintestinal sequelae suggested that Stxs damage endothelial cells. It is shown here that human brain microvascular endothelial cells (HBMECs) are relatively resistant to purified Stxs (50% cytotoxic doses [CD50s] >/=10 microgram/mL). Pretreatment of HBMECs with tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, n-butyric acid, or a cAMP analogue resulted in a 103- to 104-fold decrease in CD50 values and a 2- to 4-fold increase in fluoresceinated Stx binding to HBMECs. Treatment of HBMECs with lipopolysaccharides did not significantly alter cytotoxicity or toxin binding. TNF-alpha and IL-1beta treatment was associated with the increased HBMEC expression of the toxin-binding glycolipid globotriaosylceramide. HBMECs did not produce IL-1beta and produced only trace amounts of TNF-alpha when stimulated with purified Stx1 in vitro. (+info)
Cyclic AMP is involved in cardioregulation by multiple neuropeptides encoded on the FMRFamide gene.
(48/1207)
We have used a combination of biochemical and pharmacological techniques to investigate the role of the cyclic nucleotides, 3', 5'-cyclic adenosine monophosphate (cyclic AMP) and 3',5'-cyclic guanosine monophosphate (cyclic GMP), in mediating the cardioregulatory effects of FMRFamide and other neuropeptides encoded on exon II of the FMRFamide gene of Lymnaea stagnalis. The 'isoleucine' peptides (EFLRIamide and pQFYRIamide) produced complex biphasic effects on the frequency, force of contraction and tonus of the isolated heart of L. stagnalis, which were dependent on adenylate cyclase (AC) activity of the heart tissue. At a control rate of cyclic AMP production of less than or equal to 10 pmoles min(-)(1 )mg(-)(1) protein, the 'isoleucine' peptides produced a significant increase in AC activity in heart membrane preparations. This suggested that the enhanced AC activity is responsible for the stimulatory effects of the 'isoleucine' peptides on frequency and force of contraction of heart beat. This excitation sometimes followed an initial 'inhibitory phase' where the frequency of beat, force of contraction and tonus of the heart were reduced by the 'isoleucine' peptides. Hearts that showed the inhibitory phase of the 'isoleucine' response, but characteristically lacked the delayed excitatory phase, were found to have high levels of membrane AC activity (breve)10 pmoles min(-)(1 )mg(-)(1) protein in controls. Application of the 'isoleucine' peptides to membrane homogenate preparation from these hearts failed to increase AC activity. The addition of FMRFamide produced significant increases in the rate of cyclic AMP production in the heart membrane preparations, which could account, at least in part, for the cardioexcitatory effects of this peptide in the isolated whole heart. A membrane-permeable cyclic AMP analogue (8-bromo-cyclic AMP) and an AC activator (forskolin) were also cardioexcitatory. The peptide SEEPLY had no effects on the beat properties of the isolated heart and did not alter AC activity. The activity of the membrane-bound (particulate) guanylate cyclase (GC) was not significantly affected by any of the peptides. (+info)